**2. Current innovative strategies for microbial decontamination in winemaking**

At present, the main strategy applied to control spoilage microorganisms along the winemaking process is the addition of sulfur dioxide (SO2), a compound which is able to ensure antioxidant protection and microbiological stability. Although SO2 is a highly effective and inexpensive preservative widely used in the wine industry, concerns have been raised regarding its potentially adverse effects on human health. The general trend in the wine industry is thus currently to reduce SO2 content, or even to eliminate it altogether [7].

Dimethyl dicarbonate (DMDC), lysozyme, and sorbic acid are chemical compounds proposed as alternatives to SO2, and they are already allowed as antimicrobials in winemaking by the OIV. Although they have proven effective against certain wine spoilage microorganisms, at their maximum permitted doses none of them is sufficiently effective against the entire range of microorganisms of concern [7].

Microfiltration, on the other hand, is a common physical procedure applied in winemaking for purposes of microbial stabilization. However, this technique is only applied before bottling and has some drawbacks due to its potentially deleterious effects on flavor and color properties of wines, depending on filter media and intrinsic wine characteristics. Sterile filtration presents further practical problems associated with frequent fouling, the high cost of filters, their management, and the possible recontamination of wines during bottling [8]. Heat treatments, despite their well-known high efficacy in terms of microbial inactivation, are not commonly used in wineries due to the negative effects of high temperature on the valuable sensory properties of wine [9]. Generally, thermal pasteurization is only applied to low-medium quality wines prior to bottling.

*Microbial Decontamination by Pulsed Electric Fields (PEF) in Winemaking DOI: http://dx.doi.org/10.5772/intechopen.101112*

Similarly, emerging preservation techniques have been proposed for the microbial stabilization of wines. High hydrostatic pressure (HHP) is one of the most widely studied methods, and it has proven effective against most of the target microorganisms in wine [10]. However, due to the necessity of treating bottled wine and the possible acceleration of unwanted chemical reactions, along with the high cost and small flexibility of HPP devices, is ultimately not the most feasible technique for wineries [11]. Ultrasound, ultraviolet light, ionizing radiation, ultra-high pressure homogenization (UHPH), and microwaves have been also investigated for wine, for must, and even for barrel sterilization [12–17]. The main recent studies have focused on these techniques' lethal efficacy, but it is still necessary to obtain further knowledge about their effects on sensory quality and their actual feasibility at an industrial scale. Moreover, none of these innovative physical technologies is yet approved for wine stabilization by the OIV, except for HHP and UHPH.

In order to meet consumer demands, the wine industry is thus attempting to find new strategies to reduce or eliminate the use of SO2. However, the chosen alternative technique should ensure that the levels of inactivation required for stabilization are achieved in each step of the winemaking process, without any detectable effect on sensorial and physicochemical properties of wine.

## **3. Fundamentals of pulsed electric fields technology**

During processing with pulsed electric fields (PEF), products are subjected to very short pulses (μs) of high voltage (kV). The applied external voltage generates an electric field which, if intense enough, causes an electrical breakdown of the cell's cytoplasmic membrane. This phenomenon, referred to as electroporation, may cause the inactivation of vegetative cells of microorganisms, among other effects. The capability of PEF to inactivate microorganisms at temperatures that do not affect the flavor, color, or nutrient value of foods is highly attractive for the food industry.

#### **3.1 Principles of PEF processing**

PEF processing involves the intermittent application of direct-current voltage pulses (kV) for very short periods through a material placed between two electrodes. A typical PEF setup for food processing therefore includes a charging unit, an energy storage unit, and a switching unit that triggers pulse formation and releases the electrical pulses in the treatment chamber (**Figure 1**) [18]. According to the triggering system used for discharging the stored energy, the shape of the pulses delivered in the treatment chamber is either exponential or square. A PEF treatment chamber is composed of two electrodes held in position by insulating material, which forms an enclosure to contain the product to be treated. Parallel electrode and collinear configuration are the two proposed designs for the microbial decontamination of liquid foods by PEF [19]. Parallel electrode configuration hinders the formation of a uniform electric field in the treatment zone, whereas in a collinear treatment chamber the distribution of the electric field in the treatment zone is inhomogeneous. Nevertheless, the collinear chamber's higher load resistance, the configuration's overall lower energy requirements, and the circular section similar to the pipes used in food processing plants are nevertheless the reasons why collinear chambers are the ones currently used in industrial applications.

The effectiveness of PEF processing depends on several parameters, among which the ones most often used to describe the intensity of an applied PEF treatment are: electric field strength, processing time, total specific energy input, and

**Figure 1.**

*Simplified diagram of an electrical circuit of a PEF generator. The different pulse shapes (exponential or square) and chamber geometries (parallel and collinear electrodes) used for the application of PEF treatments in continuous conditions are plotted. The main processing parameters of PEF technology are shown below.*

temperature (**Figure 1**). Electric field strength depends on the external voltage applied, as well as on the distance between the electrodes. Treatment time represents the product's exposure time to the electric field, and depends on the number of applied pulses as well as on the pulse width. The treatment's specific energy (energy applied per mass unit) is dependent on the applied voltage, the pulse width, the number of pulses and the treatment chamber's resistance. Treatment chamber resistance varies according to its geometry and the product's conductivity. Finally, temperature is the other parameter to be considered in the evaluation of the efficiency of PEF processing in microbial inactivation. Inactivation usually increases at a higher temperature of the treatment medium – even within temperature ranges that are not otherwise lethal for microorganisms [20].

#### **3.2 Effects of an external electric field on microorganisms**

After the application of a PEF treatment, the presence of nucleic acid, proteins, and other components of the microbial cytoplasm such as adenosine triphosphate (ATP) has been observed in the medium surrounding the microorganisms. These

*Microbial Decontamination by Pulsed Electric Fields (PEF) in Winemaking DOI: http://dx.doi.org/10.5772/intechopen.101112*

observations suggest that PEF causes the formation of local defects or pores (electroporation), thereby leading to an increment of cell membrane permeability. Depending on the intensity of the treatment applied (electric field strength, processing time, specific energy) and cell characteristics (size, shape, orientation within the electric field), the electroporation of the cytoplasmic membrane can be either reversible or irreversible. It is reversible if the bilayer returns spontaneously to its initial state by recovering membrane integrity. If structural changes in the lipid bilayer due to PEF treatment are permanent, electroporation is irreversible. Permanent electroporation causes uncontrolled molecular transport across the membrane, hinders the cells' homeostatic capacity, and eventually leads to microbial death.

The electroporation of the cytoplasmic membrane caused by PEF indicates that this technology could be an effective procedure the inactivation of vegetative bacteria cells. But bacterial spores, which are a resting stage of some bacteria such as *Bacillus* and *Clostridium*, are resistant to these treatments. The low water content and unique cellular structure of bacterial spores, consisting of several layers surrounding the core, seem to provide resistance to the effect of the external highintensity electric field generated during PEF processing.
